Early endosomal, autophagic, and lysosomal systems (EALS) abnormalities and elevation of cathepsin synthesis are observed at early stages of Alzheimer's disease (AD) and Trisomy 21 (Ts21; Down syndrome). While emerging evidence show that cathepsins play an important role in initiating and mediating cell death, very little is known about in vivo regulation of cathepsins by endogenous inhibitors. Data show that the cysteine protease inhibitor cystatin C (CysC) has a protective role in brain injury and is involved in the early stages of AD. We propose to define the participation of CysC in the early and progressive activation and disruption of the EALS and to test the hypothesis that while EALS dysfunction and cathepsins elevation in AD promotes neurodegeneration, mobilization of CysC can alleviate specific consequences of the EALS pathologies observed in AD. The specific aims are: (1) To determine the cellular vesicles containing CysC and the spatial relationship of the inhibitor to lysosomal hydrolases (including cathepsins B and D) in a wel-lcharacterized collection of postmortem human AD and Ts21 brains; (2) To test in vivo our hypothesis that CysC plays a neuroprotective role in the pathobiology of AD-related EALS dysfunction in mouse models with endocytic and autophagic pathway alterations and determine whether CysC overexpression rescues and CysC deficiency promotes both EALS dysfunction and the aging-related neurodegenerative phenotype in each of these models; and (3) To study the molecular mechanism underlying the protective role of CysC, using cell culture paradigms varying in the nature of their EALS pathology or exposed to injury that results in abnormal activation or blockage of specific pathways within the EALS. Manipulating CysC levels, using cells derived from either CysC overexpressing or CysC knockout mice, and cells transfected with either CysC cDNA or CysC siRNA, will serve to demonstrate the protective role of CysC.